Isolation and Cultivation of Adult Rat Cardiomyocytes

Summary

Here, we present a protocol for the isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). Isolated ARVC can be used for short and long-term cultivation. The isolation and cultivation of ARVC can play a key role in developing new treatment regimens for cardiac diseases.

Abstract

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC).

The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl₂ until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells.

Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are β-myosin heavy chain (β-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.

Introduction

Adult cardiomyocytes in vivo work as an electrical syncytium based on cell-cell contacts between myocytes. In addition, they are influenced by adjacent cells like cardiac fibroblasts, endothelial cells, neurons, and inflammatory cells¹. In order to study the ability of cardiomyocytes to adapt their intracellular organization to altered load conditions, as seen during cardiac hypertrophy, which is an initial step leading to heart failure, the isolation and cultivation of adult ventricular rat cardiomyocytes (ARVC) is necessary^2,3,4. Historically, cardiomyocytes were first isolated from embryonic chick hearts^5,6. A few years later, the first isolation of terminally differentiated cardiomyocytes was described by using calcium depletion⁷. However, these adult cardiomyocytes were not calcium tolerant and could therefore not be used for functional assays. Finally, in 1976 a new protocol enabled Powell and Twist to investigate adult ventricular cardiomyocytes under physiological conditions⁸. As a first step, they isolated adult cardiomyocytes under low calcium concentrations and thereafter increased calcium to physiological concentrations in a stepwise procedure. Today, most protocols for the isolation and cultivation of adult cardiomyocytes work with this calcium protocol and use collagenase for the enzymatic digestion of the dense cell-cell contacts¹.

For a successful cultivation, fetal calf serum (FCS) or oncostatin M (OSM) is required. ARVC perform a de- and re-differentiation with extensive structural changes including sarcomere disassembly and reformation^9,10,11,12. This process is accompanied by a re-expression of fetal-type genes, like β-myosin heavy chain (β-MHC), as known from hypertrophy, and a formation of pseudopodia-like structures, also called spreading^4,11,13. Furthermore, swiprosin-1 (EFHD2), a newly identified protein, plays a major role in the process of re-differentiation of cultivated ARVC¹¹. As a result, ARVC in culture transform into widespread, polymorphic cells, which spontaneously show contractions after two to three weeks in culture^2,4,14.

Recent discoveries have revealed that cardiac re- and de-differentiation as it occurs under culture conditions mimics features seen in vivo during cardiac remodeling^10,15. Cardiac remodeling is a key process during cardiac diseases¹⁶. As cardiac diseases are still the main cause of death in industrialized societies, a better understanding of the biology of adult cardiomyocytes is important (WHO; 2015). Isolation and cultivation of ARVC can help to develop new strategies and medicines for the treatment of cardiac diseases. With this manuscript, a protocol for the isolation and cultivation of ARVC is provided. Furthermore, some critical parts of this method are highlighted in the discussion section.

Protocol

The investigation is conducted according to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996). In general, male wistar rats aged 3 to 4 months and with an average weight of 250 - 350 g are used for this protocol. One rat heart is sufficient for 20 culture dishes (1 mL per dish; inner diameter: 35 mm) with an approximate cell density of 1.5 x 10⁴ cells/1000 mm².

1. Preparation of Media and Reagents

1. Creatine-carnitine-taurine medium (CCT medium)
   NOTE: CCT medium is a complex medium based on medium 199 with the addition of creatine, carnitine, and taurine.
   1. Prepare 1 L of medium 199: add 3.6 g Hepes and mix for 1 h. Then add 655.5 mg creatine (5 mM), 395.4 mg carnitine (2 mM), and 625.5 mg taurine (5 mM). Carnitine and taurine change pH to <7. In order to inhibit the growth of any contaminating cells, e.g. endothelial cells or fibroblasts, add 10 µM cytosine β-D-arabinofuranoside to the medium. Adjust the pH with NaOH (2 mM) to 7.4 and sterile filter the medium. Store the CCT medium at 4 °C.
2. Powell medium
   1. For 1 L Powell medium, dissolve 6.43 g NaCl (110 mM) with 0.19 g KCl (2.5 mM), 0.16 g KH₂PO₄ (1.2 mM), 0.3 g MgSO₄ 7H₂O (1.2 mM), 5.96 g Hepes (25 mM), and 1.98 g D(+)-Glucose monohydrate (10 mM) in Aqua sterile. Adjust pH with NaOH (2 M) to 7.4 and sterile filter the medium. Store Powell medium at 4 °C.
3. Calcium chloride (CaCl₂)
   1. Prepare a 100 mM CaCl₂ solution (50 mL) and prepare aliquots containing 500 µL CaCl₂. Freeze aliquots at -20 °C.
4. Preparation of culture medium
   1. Prepare three cell culture mediums: pre-plating medium, plating medium, and washing medium. Use CCT medium as the basis for all three mediums (Table 1). Calculate CCT medium with 1 mL per culture dish. Therefore, prepare 20 mL CCT medium for 20 culture dishes (inner diameter: 35 mm).
   2. Cell culture plates: Coat each cell culture plate (inner diameter: 35 mm) with 1 mL pre-plating medium. Store the coated plates at 37 °C overnight or for at least 2 h before using.

2. Isolation of Adult Cardiomyocytes

1. Preparation of Langendorff perfusion system
   1. Heat plating medium and washing medium to 37 °C. Defreeze a tube of 500 µL CaCl₂ and weigh in 25 mg of collagenase.
   2. Flush the Langendorff perfusion system with aqua sterile, afterwards let Powell medium circulate the system for 5 min.
   3. Fill the Langendorff perfusion system with 80 mL Powell medium without any air bubbles and gas the medium with 95% oxygen.
   4. Prepare a tube (50 mL) with 40 mL Powell medium, heat it to 37 °C, and gas it with 95% oxygen.
   5. Prepare a thread of about 25 cm in length for attaching the removed heart to the cannula.
   6. Degrease a razor blade with alcohol (70% by volume) and fasten it to the chopper. Clamp a plastic disc into the chopper.
2. Extraction of heart
   1. Anesthetize a male wistar rat with 4% to 5% isoflurane and sacrifice it with cervical dislocation. Open the abdomen behind the costal arch with an abdominal shear and, with the same pair of scissors, cut through the diaphragm to open the thoracic cavity.
   2. Remove the heart, together with the lung and thymus, by cutting above the thymus highly cranial in the thoracic cavity. Transfer the material to ice-cold saline solution immediately.
   3. Remove the lung and thymus from the heart with a dissecting scissor (large) and by fixating the material with capsule forceps, transfer the latter to a new saline solution.
3. Isolation
   1. Remove excess tissue, like residues of thymus, trachea, fat, and connective tissue from the heart using capsule forceps and a dissecting scissor (large or small). Uncover the aorta and sever it with a dissecting scissor (large or small) between the first and second branchial arch.
   2. Start the dripping of the Langendorff perfusion system. Place the heart on the cannula of the Langendorff perfusion system and fixate it first with a crocodile clamp and later with the prepared thread. Rinse the heart until it is free of blood.
   3. Dissolve 25 mg Collagenase in 5 - 6 mL warm Powell medium and add 12.5 µL CaCl₂ (30 µM).
   4. Close circulation by moving a glass funnel, which is connected with the Langendorff perfusion system, over the dripping heart and add the solved collagenase to the perfusion system. Start the perfusion for 25 min with a drop velocity of 1 drop per second.
      NOTE: During perfusion, the heart will swell and get a waxy appearance.
   5. Stop the perfusion after 25 min and remove the heart from the Langendorff perfusion system. Remove the aorta, atria, and connective tissue from the heart and open the right and left ventricles.
   6. Chop the heart two times at an angle of 90° (cutting width: 0.7 mm; velocity: 0.15 cm/s). Repeat this process manually with two scalpels for 10 s each side.
   7. Transfer 12 mL of the perfusion medium into a new tube (50 mL). Pour the cell slurry into this medium and digest cells for another five minutes at 37 °C. Mix the solution every minute.
   8. Filter the solution with the digested heart through a nylon mesh (200 µm) into a new tube (50 mL).
   9. Centrifuge the filtered solution at 29 x g for 3 min. Discard the supernatant and add 6 mL warm Powell medium including 12.5 µL CaCl₂ (250 µM) to the cell pellet. Resuspend the pellet through smooth shaking movements. Centrifuge again at 29 x g for 2 min. Discard the supernatant and add 6 mL warm Powell medium substituted with 25 µL CaCl₂ (500 µM). Dissolve the cell pellet through gentle shaking movements and add 12 mL warm Powell medium including 120 µL CaCl₂ (1 mM). Centrifuge for a third time at 16 x g for 1 min. Again, remove the supernatant.
   10. Mix the cell pellet with the pre-warmed plating medium.
   11. Remove pre-plating medium from culture plates. Transfer 1 mL plating medium, including the isolated cardiomyocytes, to each culture plate. Incubate fresh isolated cardiomyocytes at 37 °C for 1 h.
   12. Remove the plating medium from culture plates. Add 1 mL washing medium to each culture plate and store the plates at 37 °C up to six days without changing the medium.
   13. For investigating the influence of different chemicals and treatments on ARVC, first refresh plating medium by washing medium and thereafter adding different chemicals.
       NOTE: Evaluation with light microscopy: With each cell preparation, 150 to 300 cardiomyocytes should be monitored per day by light microscopy. Subdivide all counted cardiomyocytes into groups according to their appearance (e.g., "rod-shaped", "round down", "spreading", and "unusual appearance"). The category "spreading" includes all cardiomyocytes with pseudopodia-like structures. "Unusual appearance" includes all ARVC with an irregular surface and no detectable intact cell membrane.

3. Example Experiments

1. Fluorescence/Immunofluorescence staining of adult cardiomyocytes
   1. Analyze morphological and structural conversions of ARVC during cultivation by confocal laser microscopy. Use Phalloidin-TRITC to investigate F-actin structures in "rod-shaped", "round down", and "spreading" ARVC. Perform staining according to the manufacturer's protocol. An example for Phalloidin-TRITC staining is given in reference Nippert et al.¹¹. With fluorescence/immunofluorescence staining, differences in the de- and re-differentiation of the contractile apparatus in cultivated adult cardiomyocytes between experimental treatments (e.g., with Swiprosin-1, ionomycin) can be investigated.
2. Real-time quantitative RT-PCR (qRT-PCR)
   1. Perform qRT-PCR to investigate changes in the mRNA expression of different genes (e.g., OSM, Swiprosin-1, β-MHC) during cultivation of ARVC. For sufficient sample size, use larger culture dishes (inner diameter: 60 mm) with 2 mL volume. ARVC of five culture dishes yields one sample. Perform isolation of mRNA and transformation of cDNA according to the manufacturer's protocol.
3. Immunoblot techniques
   1. Perform Western Blots to investigate changes in protein expression (e.g., for Swiprosin-1) during cultivation of ARVC. Use one culture dish (1 mL) per sample.

Results

Adult cardiomyocytes in culture: Figure 1 shows an overview of freshly isolated adult cardiomyocytes 2 h after the last washing. Approximately 75% of all cardiomyocytes had a rod-shaped morphology. The remaining 25% showed an unusual appearance with a round morphology and no detectable intact cell membrane (Figure 1). At the end of cultivation (day 6), up to 15% of all cardiomyocytes showed spreading, about 10% r...

Discussion

The behavior of adult cardiomyocytes in vivo is influenced by many interactions with other cells (e.g., neurons, endothelial cells, fibroblasts, inflammatory cells) and the electrical syncytium which they form¹. Therefore, studying stress adaptation of adult cardiomyocytes exclusively requires the isolation and cultivation of ARVC. The main effects of isolating and cultivating ARVC are: 1) disconnecting them from extracellular matrix and cell-cell contacts; 2) disconnecting them ...

Materials

Name	Company	Catalog Number	Comments
Langendorff perfusion system	inhouse construction		double-walled with a water based heating system
Tissue chopper Mc Ilwain	Cavey Laboratory Engeneering Co. Ltd.
Aortic Cannula, OD 1,8 mm	inhouse construction
Abdominal shears	Aeskulap	BC772R
Capsule forceps	Eickemeyer	171307
Dissecting scissor large	Aeskulap	BC562R
Dissecting scissor small	Aeskulap	BC163R
Mash with Polyamid	Neolab	4-1413	mash size 200 μm
plastic disc	Cavey Laboratory Engeneering Co. Ltd.
Collagenase Typ II	Worthington	LS004177